Renewed support for the study of structure as related to catalysis using the enzyme 2-keto-3-deoxygluconate6P aldolase of Ps. putida is requested. Experiments will be carried out to determine whether Glu-56, labeled by bromopyruvate, reductively cross-links Lys-144, the Schiff's base-forming lysine. Also experiments will ask whether this reductive cross-linking is inter or intra subunit. Further experiments will be carried out in which the Gamma-carboxylate of Glu-56 is converted to the hydroxymate. This chemically mutated enzyme species will be tested as to whether it can form the ketimine with substrates while catalytic ketimine/eneamine turnover is disallowed. Such studies will implicate Glu-56 in acid/base catalysis. In addition, alkylation of Glu-56 will be attempted with an analog of GaP. This would confirm the single-base mechanism which implicates Glu-56 participation in proton activations necessary for pyruvate and/or KDPG turnover catalyzed by the enzyme. Other experiments will probe the enzyme's kinetic mechanism in the attempt to determine the rate limiting step(s) in catalysis. Coupled to these studies will be experiments designed to reveal the pKs pertinent to catalysis, viz. carbinolamine/ketimine (complex) formation and ketimine/eneamine (catalysis) interconversion. The studies will reveal the role of the bromopyruvate-sensitive Glu-56 in catalysis, establish acid/base participation in C-C synthesis, and contribute to concepts of rational drug design.